Process and apparatus for the production of BI-212 and a use thereof

ABSTRACT

A process for producing substantially radio-impurity-free Bi-212 is disclosed. An acidic feed solution containing Pb-212 is contacted with an extraction medium to bind the Pb-212 thereto. The extraction medium is rinsed with a second acid solution to remove impurities therefrom, leaving a substantially impurity-free Pb-212-laden extraction medium The Pb-212 grows on the extraction medium to form Bi-212 by radioactive decay. The Bi-212 is then eluted from the extraction medium with an acid solution to form a substantially radio-impurity-free Bi-212 acid solution. An apparatus for carving out this process and a process for the therapeutic use thereof are also disclosed.

This application claims the benefit of U.S. Provisional application Ser.No. 60/024,567, filed Aug. 26, 1996.

DESCRIPTION

1. Technical Field

This invention relates to a process and apparatus for the production ofbismuth-212 (Bi-212) and a use for Bi-212. More particularly, theinvention relates to a process and apparatus for the production ofsubstantially radio-impurity-free bismuth-212 from a starting materialcontaining lead-212 and a therapeutic use for the bismuth-212.

2. Background of the Invention

Ovarian carcinoma has the highest mortality rate of any gynecologiccancer. This is due, in part, to the spread of the disease outside ofthe pelvis by the time the disease is diagnosed. Cytoreductive surgeryand therapy have improved the overall survival rate of patients withovarian carcinoma. However, relapses have been observed even afterapparent complete remission.

Initial treatment of patients whose cancers have reached stages III andIV with multiple chemotherapy agents yields positive responses in about90 percent of the patients. However, after four years, only about 30percent of the patients are expected to survive. Thigpen et al., Semin.Oncol., 16(Suppl. 6):58 (1989). Current treatment strategies followingrelapse include intraperitoneal chemotherapy and abdominopelvic externalbeam therapy. These treatments have usually been found to beineffective.

Radiation therapy, such as X-ray therapy, has been observed to be themost effective treatment for microscopic disease. Microscopic diseaserefers to the layers of cells remaining after removal of a tumor, cellsof a tumor that are beginning to form and the first few cell layers oftumor growth and formation. The use of radiation therapy is limited tothe radio tolerance of normal cells and by technical problemsencountered in delivering tumoricidal doses.

In addition, it is believed that when the tumor does not respond toconventional radiation therapy this may be due, in part, to the qualityof radiation that is used. For example, X-ray therapy is low-LET (linearenergy transfer), is sparsely ionizing and its effectiveness isdependent on cellular oxygen.

Radionuclide therapy using chromic phosphate (P-32), which is a low-LETbeta-emitter, has exhibited some level of success. A five-year survivalrate of 81 percent for the treatment of microscopic disease has beenreported for patients with stage I and stage II disease. Young et al.,N. Eng. J. Med., 322:1021 (1990). Nevertheless, similar to X-raytherapy, P-32 is low-LET, is sparsely ionizing and its effectiveness isdependent on cellular oxygen.

Alpha-emitting radionuclides have also been found to be effective in thetreatment and eradication of microscopic carcinoma in animal models.This is believed to be a result of the densely ionizing radiation thatis emitted during alpha-decay, and the cellular oxygen independence ofthe affect of an alpha particle on the disease.

It has been shown that lead-212 (Pb-212) and astatine-211 (At-211) areeffective in the treatment and eradication of microscopic carcinoma. Theeffectiveness of Pb-212 in treating the carcinoma is due to itssubsequent decay to Bi-212, which is an alpha-emitting radionuclide.Pb-212, itself, is not as effective as the alpha-emitting Bi-212radionuclide.

Known processes for producing alpha particle-emitting nuclides such asAt-211 are limited in that they generally require the use of particleaccelerators for production of the nuclides. Moreover, the radionuclidesso produced are often contaminated with radio-impurities that aredifficult to filter out or otherwise remove from a desired nuclide. Ithas also been found that such nuclides that are administeredintraperitoneally using a complexing agent such as Pb-212/ferroushydroxide do not have the desired property of even distribution.

Bismuth-212, which as noted above, is an alpha-emitting radionuclide,has recently been found to exhibit the desirable properties associatedwith At-211 in providing highly ionizing radiation and exhibitingcellular oxygen independence. Moreover, certain formulations of Bi-212made in accordance with this invention as discussed hereinafter havealso been found to overcome the distributional problems encountered withcomplexed Pb-212 and At-211 upon intraperitoneal administration. Inaddition, Bi-212 has a half-life of 60.6 minutes, which makes thisisotope useful for intraperitoneal treatment because it emits itsradiation while its distribution in the peritoneal fluid is uniform.

Nevertheless, problems have been encountered in the production ofBi-212. The production of Bi-212 is dependent upon natural radioactivedecay, and impurities are typically present in the Bi-212 final product.Bi-212 is produced from a Thorium-228 (Th-228) source. The decay ofTh-228 produces radium-224 (Ra-224) which decays to Radon-220 (Rn-220),which decays to Polonium-216 (Po-216), which decays to Pb-212, which inturn decays to Bi-212. Those skilled in the art will recognize thatimpurities (e.g.; the parent isotopes and daughter isotopes of Bi--212)can adversely affect treatment of and eradication of the carcinoma.

Moreover, because of the highly ionizing nature of the parent isotopesthat decay to Bi-212 and in particular, the alpha-emitting isotopes, andbecause of the relatively short half-life of Bi-212, it would be moredesirable to produce Bi-212 at a location remote from a Th-228 source,and as physically close to the patient as possible. It would, of course,be most beneficial to produce the isotope at the patient's "bed-side" toreduce the stress on the patient and reduce or eliminate the need forspecifically designed facilities for radiotherapy.

Accordingly, there continues to be a need for a process and apparatusfor the production of Bi-212 that is substantially free ofradio-impurities. There is also a need for a process and apparatus thatpermit local production of Bi-212 at a location remote from theassociated primary Th-228 source. Such an apparatus should further besufficiently portable so it can be transported to a patient foradministration of and treatment with Bi-212 without the need for specialfacilities such as intensive radiation shielding. The disclosure thatfollows provides one such apparatus and a method or process for its use,as well as a therapeutic process for using the Bi-212 so prepared.

SUMMARY OF THE INVENTION

A process of producing substantially radio-impurity-free Bi-212 iscontemplated. That process comprises the steps of contacting an acidicPb-212 feed solution containing Pb-212 or a Pb-212-generating materialwith an extraction medium having a plurality of high affinity Pb-212binding sites thereon, to form a Pb-212-laden extraction medium that cancontain contaminants. The Pb-212-laden extraction medium is rinsed witha second acid solution to remove contaminants therefrom and form asubstantially impurity-free Pb-212-laden extraction medium. The Pb-212on the extraction medium is incubated (maintained) for a predeterminedperiod of time so as to form Bi-212 from the Pb-212 by radioactivedecay. A third acid solution is introduced to the Pb-212-ladenextraction medium to release the Bi-212 therefrom, and form an acidsolution containing Bi-212. The solution is eluted from theimpurity-free Pb-212-laden extraction medium to form a substantiallyradio-impurity-free Bi-212 acid solution. That acid solution can besubsequently neutralized for administration to a patient.

In a preferred process, a first acid solution is introduced to astarting material having Pb-212 or a Pb-212-generating material to formthe acidic Pb-212 feed solution.

In another preferred embodiment, the substantially radio-impurity-freeBi-212 acid solution that is eluted from the extraction medium iscontacted with a subsequent extraction medium also having high Pb-212affinity characteristics to remove Pb-212 that can break through fromthe first extraction medium contact. In a most preferred process, thefirst and second acid solutions are hydrochloric acid in concentrationsof about 0.9 N to about 2.0 N.

The substantially radio-impurity-free Bi-212 acid solution can beneutralized with, for example, sodium hydroxide (NaOH) or any otherpharmaceutically acceptable base and diluted to form an isotonicsolution for patient administration.

An apparatus for producing substantially radio-impurity-free Bi-212 froma starting material of Pb-212 or a Pb-212 generating material includesan extraction medium having an affinity for binding Pb-212 thereto and alower affinity for binding Pi-212 thereto. A first acid supply is inflow communication with the starting material and is adapted to supply afirst acid solution to carry the Pb-212 to the extraction medium. Theapparatus includes a first vessel adapted to retain the extractionmedium and to maintain contact between the extraction medium and thefirst acid. A second acid solution supply is in flow communication withthe vessel and is adapted to supply a second acid thereto.

The apparatus includes a mixing chamber in flow communication with thevessel adapted to receive a liquid solution from the vessel. The mixingchamber includes a plurality of inputs for adding solutions to themixing chamber to, for example, neutralize and dilute the solutiontherein. A discharge line is in flow communication with the mixingchamber for discharging liquid therefrom.

A preferred embodiment of the apparatus includes a second vesselpositioned between the first vessel and the mixing chamber. The secondvessel is loaded with an extraction medium that also has high Pb-212affinity characteristics and low Bi-212 affinity characteristics.

The present process and apparatus facilitate the production ofsubstantially radio-impurity-free Bi-212, and the preparation of aBi-212 solution for patient administration. The apparatus can beconfigured for transport to a patient and for local production of Bi-212for rapid administration. The present process and apparatus remove theconstraints of known processes and apparatuses, particularly relating tothe production of the radio-nuclide and the purification thereof.

A process for treating target cells includes contacting target cellswith a biologically effective amount of a pharmaceutically acceptablecomposition that comprises an aqueous suspension of substantiallyradio-impurity-free Bi-212. In a preferred process, the Bi-212 isuncomplexed. The target cells can be of microscopic carcinoma and theBi-212 suspension can be administered to a host mammal in need thereofintrapertioneally.

Advantageously, the present process for producing Bi-212 produces asubstantially radio-impurity-free Bi-212 acid solution that is free ofcontamination from Bi-212 parent radionuclides. Thus, although Bi-212decay products exist in the solution because of the decay of Bi-212, thelevel of other radioactive nuclide present is not measurable.

An apparatus for producing the substantially radio-impurity-free Bi-212acid solution is sufficiently compact that the apparatus can be readilytransported to and from a patient's bed-side without special facilities,such as extensive shielding. The apparatus is contained in a relativelycompact lead-shielded container that permits ready transportation.Moreover, the apparatus permits the production of Bi-212 remote from thebefore-noted Th-228 source.

The substantially radio-impurity-free Bi-212 that is produced can beused in a variety of therapeutic applications. Adventageously, theBi-212 that has been prepared for therapeutic use distributes evenlywithin the peritoneal fluid during the time that the Bi-212 "delivers"its radiation to target cells. That is, unlike known alpha-emittingpreparations such as Pb-212/ferrous hydroxide, which "clump" within theperitoneal cavity, a contemplated Bi-212 preparation distributes evenlyto contact target cells within the host mammal.

Other features and advantages of the present invention will be apparentfrom the following detailed description, the accompanying drawings, andthe appended claims.

BRIEF DESCRIPTION OF THE FIGURES

In the figures forming a portion of this disclosure,

FIG. 1 is a simplified flow diagram of a process for the production ofrelatively radio-impurity-free Bi-212, embodying the principles of thepresent invention;

FIG. 2 is a schematic arrangement of an apparatus for the production ofBi-212;

FIG. 3 is a graphic illustration of the decay rate of Pb-212 (triangles)in milliCuries (mCi) relative to the production rate of Bi-212(circles);

FIG. 4 is an illustration of the Th-228 decay chain showing the decayproducts thereof including radium-224, lead-212 and bismuth-212, thedecay process (alpha or beta) and half-lives of the decay products;

FIG. 5 is a graphic illustion of the percent of cells surviving as afunction of the radiation dose received in Greys (Gy) of monolayer cellsof V-79 subjected to X-ray radiation (open hexagons), V-79 cellssubjected to P-32 chromic phosphate (open squares), V-79 cells subjectedto Bi-212 (open triangles), Ehrlich-Lettre Ascites carcinoma cellssubjected to Bi-212 (open diamonds), OVCAR-3 cells subjected to X-rayradiation (open circles), OVCAR-3 cells subjected to P-32 chromicphosphate (filled circles) and OVCAR-3 cells subjected to Bi-212(filled, inverted triangles);

FIG. 6 is a graphic illustration of the percent of cells surviving as afunction of the radiation dose received in Gy of 800 μm spheroids ofOVCAR-3 cells that were subjected to X-ray radiation (filled, invertedtriangles), P-32 chromic phosphate (open squares) and Bi-212 (opencircles); and

FIG. 7 is a graphic illustration of the percent of mice that survived,as a function of days following injection, that were injected with 10⁶Ehrlich-Lettre Ascites carcinoma cells (filled circles) and mice thatwere injected with 10⁶ carcinoma cells and subsequently treated with 100μCi of Bi-212.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although the present invention is susceptible of embodiment in variousforms, there is shown in the drawings and will hereinafter be describeda presently preferred process and a presently preferred embodiment of anapparatus with the understanding that the present disclosure is to beconsidered an exemplification of the invention and is not intended tolimit the invention to the specific process and apparatus illustrated.

Referring now to the figures and particularly to FIG. 1, there is showna simplified flow diagram depicting a process for producingsubstantially radio-impurity-free Bi-212. Bi-212 is produced from astarting material containing lead-212 (Pb-212) or a Pb-212-generatingmaterial such as radium-224 (Ra-224). The starting material can beprovided, for example, in accordance with the teachings of U.S. Pat. No.4,663,129 to Atcher et al., whose disclosures are incorporated herein byreference.

In the present process, a first acid solution is introduced to thestarting material to a form a Pb-212 feed solution. The Pb-212 feedsolution is contacted with an extraction medium having a plurality ofbinding sites thereon adapted to bind the Pb-212 thereto, and form aPb-212-laden extraction medium and less strongly bound contaminants. ThePb-212-laden extraction medium is rinsed with a second acid solution toremove the contaminants therefrom and to form a substantiallyradio-impurity-free Pb-212-laden extraction medium.

The impurity-free Pb-212-laden extraction medium is incubated (ismaintained) for a predetermined period of time so as to form Bi-212 fromPb-212 by radioactive decay as illustrated in FIG. 3. This type ofmaintenance step is often referred to in the art as "growing" a desiredradionuclide. A third acid solution is introduced to the extractionmedium to release the Bi-212 therefrom, to form a Bi-212 acid solution.The Bi-212 acid solution is then eluted from the Pb-212-laden extractionmedium. The eluant is a substantially radio-impurity-free Bi-212 acidsolution; i.e., the solution contains Bi-212 and its decay products, butis free of decay products from radionuclides other than Bi-212. That is,the resulting solution is greater than 95 percent radio-impurity-free,and more preferably greater than 99 percent radio-impurity-free.

Bi-212 is produced from Pb-212 by radioactive decay. As shown in FIG. 3,Bi-212 is a daughter product of Pb-212. Pb-212 has a half-life of about10.6 hours, and decays to Bi-212 through beta decay. The Pb-212 ispurified to remove any radio-impurities that may be present, and ispermitted to decay to produce Bi-212. The Bi-212 is then purified toform a relatively radio-impurity free stream of Bi-212.

An acid solution is used to transfer the Pb-212 from a source, such as aradium generator as described in the aforementioned Atcher patent, tothe extraction medium. In a present embodiment, the extraction medium isa solid phase-supported (e.g., resin-supported) extractant, referred toas an extraction chromatographic resin. The extraction medium has aplurality of binding sites that have a relatively high affinity for ionsof Pb-212 and a lower affinity for ions of Bi-212, as well as ions ofisotopes of thorium and radium, such as Th-228 and Ra-224.

In the current embodiment, the column is loaded with "Sr Resin™", ananalytical resin available from Eichrom Industries, Inc. of Darien,Ill., that is described in U.S. Pat. No. 5,110,474, which disclosure isincorporated by reference. Briefly, the Sr Resin comprises an inertresin substrate upon which is dispersed a solution of the extractant,namely, a crown either dissolved in a liquid diluent.

The diluent is an organic compound that has (i) a high boiling point;i.e. about 1700 to 200° C., (ii) limited or no solubility in water,(iii) is capable of dissolving from about 0.5 to 6.0 M water, and is amaterial (iv) in which the crown ether is soluble. These diluentsinclude alcohols, ketones, carboxylic acids and esters. Suitablealcohols include 1-octanol, which is most preferred, although 1-heptanoland 1-decanol are also satisfactory. The carboxylic acids includeoctanoic acid, which is preferred, in addition to heptanoic and hexanoicacids. Exemplary ketones include 2-hexanone and 4-methyl-2-pentanones,whereas esters include butyl acetate and amyl acetate.

The macrocyclic polyether can be any of the dicyclohexano crown etherssuch as dicyclohexano-18-Crown-6, dicyclohexano 21-Crown-7, ordicyclohexano-24-Crown-8. The preferred crown ethers have the formula:4,4' (5') [R,R')dicyclohexano]-18-Crown-6, where R and R' are one ormore members selected from the group consisting of H and straight chainor branched alkyls containing 1 to 12 carbons. Examples include, methyl,propyl, isobutyl, t-butyl, hexyl, and heptyl. The preferred ethersinclude dicyclohexano-18-crown-6 (DCH18C6) andbis-methylcyclohexano-18-crown-6 (DMeCH18C6). The most preferred etheris bis-4,4' (5') [(t-butyl)cyclohexano]-18-Crown-6 (Dt-BuCH18C6).

The amount of crown ether in the diluent can vary depending upon theparticular form of the ether. For example, a concentration of about 0.1to about 0.5 M of the most preferred t-butyl form (Dt-BuCH18C6) of theabove-noted preferred crown ether in the diluent is satisfactory, withabout 0.2 M being the most preferred. When the hydrogen form is used,the concentration can vary from about 0.25 to about 0.5 M.Concentrations above about 0.5 M of the crown ether in the diluent donot improve lead recovery when R and R' are H.

The preferred Sr Resin™ utilizes an inert resin substrate that is anon-ionic acrylic ester polymer bead resin such as Amberlite®XAD-7 (60percent to 70 percent by weight) having a coating layer thereon of acrown ether such as 4,4'(5')di-t-butylcyclohexane-18-crown-6(bis-t-butyl-cis-dicyclohexane-18-crown-6) (20 percent to 25 weightpercent) dissolved in n-octanol (5 percent to 20 weight percent), havingan extractant loading of 40 weight percent. E. P. Horwitz et al.,Solvent Extractions and Ion Exchange, 10(2),313-16 (1992).

It has also been observed that Pb Resin™, a related resin, alsoavailable from Eichrom Industries, is also useful for purifying andaccumulating Pb-212 for the production of Bi-212. Pb Resin™ has similarproperties to Sr Resin™ except that a higher molecular weight alcohol;i.e., isodecanol, is used in the manufacture of Pb Resins. E. P. Horwitzet al., Analytica Chimica Acta, 292, 263-73 (1994). It has been observedthat Pb Resin™ permits subsequent extraction of the Pb-212 from theresin, whereas it has been observed that Pb-212 becomes essentiallyirreversibly bound to the Sr Resin™.

It is to be noted that the present process can be carried out bycontacting the Pb-212 feed solution with a medium other than theabove-noted solid phase-supported extractant. For example, it iscontemplated that the Pb-212 feed solution can be contacted with theextractant (e.g., the crown ether dissolved in the liquid diluent) in aliquid-liquid extraction process. The extractant and exemplary processesfor the use thereof are disclosed in U.S. Pat. No. 5,100,585 to Horwitzet al., which disclosure is incorporated by reference. Such otherprocesses are within the scope of the present invention.

The Pb-212 is transferred to the resin-loaded column in an acidicelutriant solution. A preferred elutriant is hydrochloric acid (HCl)having a concentration of about 0.5 N to about 4.0 N. It has beenobserved that when Ra-224 is used as the starting material, the use ofacid concentrations above about 3.0 N can cause breakthrough of theRa-224 from the resin column. As such, the concentration of the acidicelutriant can be adjusted accordingly to prevent radium breakthroughthrough the resin column. Nitric acid has also been shown to be aneffective elutriant. Other monobasic acids, such as hydroiodic acid, canalso function effectively as elutriants.

The loaded column is then rinsed with an acid solution, such as HCl, toremove radio-impurities therefrom. Preferably, the acid rinse solutionhas a concentration of about 0.5 N to about 4.0 N, and most preferablyabout 2.0 N. The impurities present on the Pb-212-loaded resin columncan include Bi-212, Th-228 and Ra-224. Because the Sr Resin™ has ahigher affinity for Pb-212 than other radioisotopes, the impurities arerinsed from the resin by the acid solution. The acid solution containingthe impurities is forwarded from the column to a waste receptacle. Thewastes are handled and treated in accordance with good practices as willbe recognized by those skilled in the art.

After the column is rinsed, the column contains a substantiallyimpurity-free Pb-212-loaded resin complex. The Pb-212 bound to the resinis then allowed to incubate on the column to "grow" Bi-212. As will berecognized by those skilled in the art, Pb-212 has a half-life of about10.6 hours. The decay of Pb-212 produces, by beta decay, the daughterBi-212, which has a half-life of about 60.6 minutes.

As the Pb-212 decays, the concentration of Bi-212 on the resinincreases. A graphic representation of the relative decay and productionrates of Pb-212 and Bi-212 is illustrated in FIG. 3. As is apparent fromFIG. 3, the concentration of Bi-212 increases significantly at first andreaches a maximum yield at about four hours. However, after about twohours, the yield of Bi-212 is sufficiently close to the maximum yield toelute the Bi-212 from the column in a batch processing mode. It will berecognized by those skilled in the art that the present process is notlimited to a batch processing method. Rather, steady state productionand elution of Bi-212 is contemplated by the present process, and iswithin the scope of the present invention.

After the Bi-212 has been permitted to grow for a predetermined periodof time, the Bi-212 is eluted from the column using an acid solution. Apreferred acid for eluting the Bi-212 is HCl in a concentration of about0.5 N to about 1.5 N, and most preferably about 0.9 N. Other acids canbe used to elute the Bi-212, such as nitric acid, hydroiodic acid andthe like. As the acid contacts the resin, the Bi-212 is carried away bythe solution, whereas the Pb-212 remains bound to the resin. Theresulting solution contains substantially radio-impurity-free Bi-212. Inusual and most preferred practice, the solution is 99.99 percent free ofradio-impurities.

In a preferred process, the substantially radio-impurity-free Bi-212acid solution is fed into a subsequent column containing a quantity ofSr Resin™. Because of the resin's high affinity for Pb-212, any Pb-212that may have broken through from the first column is bound to andcaptured by the second column during contact of the Bi-212 acid solutionwith the resin. The Bi-212 is eluted from the resin in the second columnusing an acid solution similar to that used to elute the Bi-212 from thefirst column. The Bi-212 passes through the second resin columnunaffected and ready for preparation for patient administration.

To prepare the substantially radio-impurity-free Bi-212 acid solutionfor patient administration, the solution is transferred to aneutralizing and dilution chamber. The solution is neutralized with anappropriate amount of a base, such as sodium hydroxide (NaOH) or anotherpharmaceutically acceptable base, and is diluted to produce an isotonicBi-212-containing preparation. NaOH is a preferred neutralization agentbecause it produces a saline solution when HCl is used as the elutriant.An indicator solution, such as phenolsulfonthaline can be added to thepreparation to monitor the pH value thereof. The preparation can bediluted with deionized water to produce an isotonic preparation, e.g.,about 0.85 percent saline, for patient administration.

The Bi-212-containing preparation is assayed, prior to administration tothe patient, to determine the activity of the final product. As will berecognized by those skilled in the art, the decay of Bi-212 produces theshort lived daughter product thallium-208 (Tl-208). Tl-208 is a highenergy beta-gamma emitter having a half-life of about 3.0 minutes. Assuch, the Bi-212 is held for about 15 minutes prior to assaying, topermit the Tl-208 to reach equilibrium with the Bi-212. After assayingthe preparation, it can be administered to a patient in accordance withprinciples that will be recognized by those skilled in the art.

A schematic arrangement of an apparatus 1 that is used to carry out thepresent process is illustrated in FIG. 2. The apparatus includes asource or starting material having Pb-212 or a Pb-212 generatingmaterial. In a current embodiment, the starting material is provided bythe radium generator 10 as described in the aforementioned patent toAtcher et al.

In the illustrated embodiment, the generator 10 is in flow communicationwith a pair of acid solution storage sources 12, 14 for providingaqueous acid solutions to the generator. Flow communication is providedby tubing t, such as Tygon® tubing extending between the acid sources12, 14 and the generator 10. The storage sources 12, 14 containdifferent concentrations of, for example, HCl, at concentrations of 2.0N and 0.9 N, respectively. HCl at different concentrations, as well asother acids can be stored in the storage sources 12, 14. Valves v arepositioned in the system 1 to initiate and terminate the flow of acid tothe generator 10.

The generator 10 is also in flow communication with a first column 16,and preferably, a pair of identical "first" columns 16a. For purposes ofthe present discussion, reference will be made to a first column. It isto be understood that reference to the first column is to one of thepair of first columns.

In a preferred embodiment, the first column 16 contains a predeterminedquantity of an extraction medium 18, such as the aforementioned SrResin. As is readily apparent from FIG. 2, the apparatus 1 is configuredsuch that one of the two columns 16a can be in service while the othercolumn 16b is idle. This arrangement provides redundancy in the system1, and further permits extended use time of the system 1 by extendingthe process capacity thereof.

It is in the first column 16a, b that the Pb-212 is contacted with theextraction medium or resin 18. The Pb-212 binds with the resin 18 toform the Pb-212 laden resin. The Pb-212 laden resin is then rinsed with,for example, 2.0 N HCl to remove impurities, and is bound to the resin,to grow the Bi-212.

After a predetermined growth period, the Bi-212 is eluted from thecolumn using, for example, 0.9 N HCl from the acid source 14. As can beseen from FIG. 2, the tubing t from the acid sources 12, 14 isconfigured using three-way valves to provide acid to either thegenerator 10 or to the first column 16.

The discharge from the "first" column can be direct to either a wastereceptacle 20 or to a "second" column 22. Referring to the previouslydescribed process, the discharge from the first column 16 is asubstantially impurity-free Bi-212 acid solution.

The discharge that is directed to the second column 22, the eluant ofthe first column 16, is contacted with a similar extraction medium 24 asthat that is loaded into the first column 16. The medium 24 in thesecond column 22 thus serves to remove any Pb-212 that may have brokenthrough from the first column 16.

The discharge from the second column 22 can be directed to a clean-upcolumn 36. The clean-up column 36 can include a quantity of material toabsorb any extractant or solvent that may have been removed from the SrResin™ and carried out of the column 22. It is contemplated that anon-ionic acrylic ester polymer bead resin, such as that used to supportthe extractant in the Sr Resin™ can be used in the clean-up column 36.Alternately, the clean-up column 36 can be combined into the column 22,and can be disposed at about the bottom of the column 22 to absorb anyextractant or solvent that may be carried from the Sr Resin™.

The discharge 26 from the second column 22 is directed to a mixing andneutralization chamber 28. The chamber 28 includes various feed lines,such as a NaOH feed line 30 for providing a neutralizing agent toneutralize the acid solution, a deionized (preferably sterile andpyrogen-free) water feed line 32 for diluting the Bi-212 solution andfor rinsing the chamber 28 and an isotonic solution feed line 34 fordiluting the solution prior to patient administration.

In one embodiment, mixing is provided to the chamber 28 by gas injectionto a bubbler system 40. Alternately, a magnetic stirrer (not shown) orlike stirring device can be used to mix the solution. Essentially, thesolution in the chamber 28 is mixed by agitation provided by gas, suchas air, forced into the liquid or by, for example, mechanical agitation.The final product, ready for patient administration is dischargedthrough a discharge line 42.

The waste receptacle 20 is adapted to receive liquid and gaseous wastesgenerated during the Bi-212 production process. Due to the possibilityof generating radon-220 (Rn-220), the system 1 is configured to receiveand filter gases that can be produced during the process. The design andconfiguration of such a gaseous waste storage and processing system isnot within the scope of the present invention, and will be recognized bythose skilled in the art.

As can be seen from FIG. 2, the system 1 includes a plurality of valvesv positioned between the various liquid sources, columns and chambers todirect flow to the desired equipment to affect the desired processsteps. In a preferred embodiment, the valves are remotely actuated, suchas by solenoid actuators. This can considerably reduce radiationexposure to personnel.

The apparatus 1 can be subjected to high levels of radioactivity, and isa source of potentially high levels of radiation exposure when in use.Because of the nature of the radioactive sources used, e.g., the radiumgenerator 10, and the radioisotopes produced thereby, the apparatus 1 ispreferably housed in a radiologically shielded chamber or housing (notshown). Metallic lead is recognized as a preferred material forradiological shielding because of its high radiation attenuationproperties. Moreover, because the physical apparatus 1 can betransported to a patient, rather than transporting the patient to theapparatus 1, lead is a preferred shielding material to reduce theoverall physical size of the apparatus. In a present embodiment, thehousing has about 5 to 6 inches of lead shielding.

In a present embodiment, the apparatus 1 is carried by a sleeve (notshown) within the shielded housing. In order to maintain the integrityof the process generally, and in particular the final product, atitanium or like, highly corrosion resistant sleeve is positioned in thehousing and is configured to carry all of the process equipment,including the generator 10, the columns 16, 22, the mixing andneutralization chamber 28 and the waste receptacle 20.

As will be recognizable to be able to monitor the process both visuallyand radiologically, a periscope-like viewing port, a fiber optic viewingapparatus or like device can be positioned in the housing, or thehousing can be configured to permit insertion and withdrawal of such avisual monitoring device. In addition, it has been found that the it isdesirable to be able to radiologically monitor the mixing andneutralizing chamber 28 and the waste receptacle 20. As such, openingscan be provided within the housing, while maintaining radiologicalcontrol of the system, such that a remote radiation detector or likemonitoring device can be used to determine the radiation emitted fromthe mixing chamber 28 and the waste receptacle 20.

When operating in batch mode, to produce a maximum yield of Bi-212, ithas been found that it is most effective to elute the Bi-212 from thefirst column after an incubation or growth period of about two hours,and to elute the Bi-212 in successive intervals of about two hours. Ithas been found that a Bi-212 yield of about 79 percent of thetheoretical yield can be achieved using 0.9 N HCl as the elutingsolution. The theoretical yield of Bi-212 from the decay of Pb-2l2 isdetermined by the expression:

    Bi-212=[Pb].sub.20 (λ.sub.Bi /λ.sub.Bi -λ.sub.Pb) [exp (-λ.sub.Pb t)]-[-exp (-λ.sub.Bi t)]

where

λ_(Bi) =0.6863,

λ_(Pb) =0.0654,

λ=1n 2/t_(1/2), and

t is the elution interval.

Table 1 illustrates the theoretical yield of Bi-212 at varying elutionintervals, and at varying periods of "growth" of Bi-212.

                  TABLE 1                                                         ______________________________________                                        CALCULATED YIELD OF Bi-212 IN mCi                                             OBTAINED FROM 1 mCi OF Pb-212                                                 TIME OF GROWTH OF Pb-212                                                      ELUTION                                                                       INTERVAL (HRS)                                                                           4 HRS   8 HRS    12 HRS                                                                              24 HRS                                                                              INFINITY                              ______________________________________                                        0.25       2.219   3.928    S.243 7.635 9.642                                 0.5        2.043   3.615    4.826 7.027 8.875                                 1.0        1.741   3.082    4.113 5.990 7.565                                 2.0        1.295   2.292    3.059 4.455 5.626                                 4.0        0.780   1.380    1.842 2.683 3.388                                 ______________________________________                                    

The calculated yield of Bi-212 has been found to be dependent on theelution time interval. For example, as illustrated in Table 1, elutingthe column at fifteen (15) minute intervals over a twenty-four (24) hourperiod theoretically yields 7.635 mCi of Bi-212 for each mCi of Pb-212initially introduced to the column.

The actual yields of Bi-212 in an above-prepared radio-impurity-freesolution have been shown to vary from the calculated theoretical yieldsdepending upon the concentration of the acid solution used to elute theBi-212. As illustrated in Table 2, the percent of theoretical yield wasshown to vary between 6.9 percent and 90.0 percent for concentrations ofnitric acid between 0.1 N and 2.0 N, and between 78.0 percent and 94.0percent for concentrations of HCl between 0.5 N and 2.0 N.

                  TABLE 2                                                         ______________________________________                                        COMPARISON OF ELUTIONS WITH NITRIC                                            AND HYDROCHLORIC ACIDS                                                                   PERCENT YIELD                                                                              PERCENT YIELD                                         CONC. OF ACID                                                                            NITRIC ACID  HYDROCHLORIC ACID                                     ______________________________________                                        0.1 N      6.9                                                                0.2 N      45.6         --                                                    0.3 N      65.6         --                                                    0.5 N      70.0         78                                                    0.9 N      79.9         79                                                    2.0 N      90.0         94                                                    ______________________________________                                    

The actual yields of Bi-212 at varying elution times are shown in Table3 below. The Bi-212 was eluted using a hydrochloric acid solution at aconcentration of 0.95 N.

                  TABLE 3                                                         ______________________________________                                        ACTUAL YIELDS OF Bi-212 GENERATED FROM Pb-212                                 AT VARIOUS ELUTION TIMES                                                      Pb-212 (mCi)  ELUTION TIME                                                                              Bi-212 (mCi)                                        ______________________________________                                        37.2          1 HR. 41 MIN                                                                              20.1                                                35.3          5 HR. 23 MIN.                                                                             30                                                                2 HR. 21 MIN.                                                                             19.9                                                14.5              38 MIN.*                                                                              2.3                                                 10.1          4 HR.  7 MIN.                                                                             10.9                                                              6 HR. 40 MIN.*                                                                            11.8                                                18.5          2 HR. 55 MIN.                                                                             13.9                                                17.7          5 HR. 10 MIN.                                                                             14.2                                                              7 HR. 43 MIN.*                                                                            10.3                                                5.9           3 HR. 20 MIN.                                                                             4.9                                                 6.6           3 HR. 18 MIN.                                                                             4.5                                                 3.1           4 HR. 13 MIN.                                                                             3.8                                                 ______________________________________                                         *Bi-212 permitted to regrow after initial elution                        

The solubility of Bi-212 in a radio-impurity-free solution has beenobserved to be dependent upon the pH value of the solution, with thesolubility increasing with a decrease in pH value. The solubility wasmeasured by passing the solution through a 0.22 μ Nalgene® filter andmeasuring the activity of the resulting solution and the activity of thefilter. The results are presented in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        SOLUBILITY OF Bi-212 AT VARIOUS pH VALUES                                                     PERCENT    ACTIVITY IN                                                                            PERCENT                                        ACTIVITY ON                                                                              ACTIVITY ON                                                                              SOLUTION ACTIVITY IN                               Ph   FILTER (mCi)                                                                             FILTER     (mCi)    SOLUTION                                  ______________________________________                                        6    0.24        4         5.55     96                                        7    0.023       6         3.24     94                                        8    0.50       17         1.90     83                                        9    1.35       68         0.65     32                                        ______________________________________                                    

A substantially radio-impurity free Bi-212 solution described herein isuseful in a process described below in that such a solution caneffectively treat, e.g., cause the death of or otherwise retard thegrowth of, target cells. A particularly preferred solution includes asubstantially radio-impurity free aqueous Bi-212 preparation at pH 7.4such as that obtained by neutralizing the before-described acidic Bi-212solution. Preferably, the acidic Bi-212 solution is neutralized using1.0 N NaOH.

As the Bi-212 solution is neutralized, water-insolublebismuthoxychloride (BiOCl) is formed as a dispersion in the aqueousphase. The Bi-212 in this form is uncomplexed. That is, the Bi-212 isnot complexed with complexing agents such as antibodies,diethylenetriaminepentaacetic acid (DTPA) and the like.

Bismuthoxychloride is insoluble in water, but when formed as describedherein, a clear composition results. It is believed that the BiOCl ispresent in this composition as a colloidal dispersion or othernon-setting dispersion and the composition is referred to simply as adispersion.

In addition, while being highly effective in the treatment anderadication of carcinoma, an uncomplexed Bi-212 dispersion exhibitscharacteristics that can make it useful in other medical treatments. Ithas been observed that various radionuclides such as Iridium-192,Yttrium-90 and Rhenium-188 can be useful in the treatment of arthritisand coronary heart disease because of their radioactive properties.Raloff, Science News, 152:40-42 (1997). An uncomplexed Bi-212 dispersioncan similarly be a successful treatment preparation because of itsrelatively short half-life, and its even distribution characteristics.

In treating a disease condition as described above, a contemplatedBi-212 dispersion is administered or instilled into the body of thepatient in need thereof, e.g., the host mammal such as a rabbit, amouse, a rat, a dog, a primate such as a monkey, ape or human beingtreated, and preferably into an enclosed, cavity-like area such as intothe peritoneum or the knee joint. The dispersion can also beadministered into, for example, a coronary artery. The dispersion isadministered in a form in which the Bi-212 is present as BioCl.

In an anticipated use, the total dose administered to the patient insingle or divided doses, such as by a continual administration can be inamounts, for example, of up to about 300 mCi over a period of about 6hours.

The dosage regimen for treating a disease condition with a Bi-212dispersion is selected in accordance with a variety of factors,including the type, age, weight, sex, diet and medical condition of thepatient, the severity of the disease, pharmacological considerationssuch as the activity, efficacy, pharmacokinetic and toxicology profilesof the Bi-212 dispersion and whether the dispersion is administered aspart of a drug combination. A variety of factors specific to the type oftreatment can also be considered. For example, in the treatment anderadication of carcinoma, the configuration and size of the tumor isalso to be considered. Thus, the dosage regimen actually employed canvary widely and therefore can deviate from the preferred dosage regimenset forth above.

As discussed above, it has been observed that Bi-212, which is an alphaparticle emitter, is an effective radionuclide for use in the treatmentand eradication of microscopic carcinoma such as that diseased tissuethat arises from metastatic ovarian cancer. In a most effective use, theBi-212 is maintained in solution or as part of a non-settling dispersionduring its decay. It has been found that Bi-212 is maintained insolution at an acidic pH value.

Preferably, the substantially radio-impurity free Bi-212 acid solutionis maintained at a pH value of about 7.4 for use. It will be recognizedthat the solution, as eluted from the column, is at a lower pH valuethan 7.4. As discussed above, the addition of a neutralizing agent, suchas NaOH or another pharmaceutically acceptable base is used toneutralize the acidic Bi-212 solution to a pH value of about 7.4. Thesolution is administered with other ingredients such as sterile H₂ O toproduce an isotonic solution for administration.

Previous attempts to use alpha-emitting nuclides in treating cancerouscells were unsuccessful in that such attempts used a vehicle (such asPb-212 ferrous hydroxide) to carry the nuclide into contact with thediseased, e.g., cancerous, cells. It was observed shortly afterintroduction of ferrous hydroxide Pb-212, that the Pb-212 agglomerated,forming "clumps" resembling Dijon mustard. Thus, problems wereencountered with the distribution of such nuclides when used with thesevehicles. Similar difficulties were noted using P-32 chromic phosphate.

Unlike previous attempts to use alpha-emitting nuclides and P-32, thepresent Bi-212 dispersion is introduced into a confined cavity withinthe patient's body such as the peritoneum without an iron-based or otherion-suspending vehicle or complexing agent being required and preferablyabsent. Biological studies have shown that such an intraperitoneallyadministered Bi-212 dispersion exhibits even distribution within theperitoneal fluid. It is to be noted that the rate of decay of Bi-212 issufficiently fast (i.e., the half-life is sufficiently short) that any"clumping", if it occurs at all, happens after the Bi-212 has decayed toits daughter products and has "delivered" its radiation to the targetcells.

Biological studies were conducted to illustrate the effectiveness ofBi-212 prepared using a contemplated apparatus and process, as well asthe nuclide distribution upon introduction into the peritoneal fluid,and through the duration of the decay of Bi-212. The following examplesare intended to exemplify the invention and are not intended to limitthe invention to the specific examples described herein.

EXAMPLE 1 In Vitro Studies

In vitro studies were carried out using three well known cell lines, thecells from which were exposed to radiation from: (1) an X-ray source;(2) P-32 chromic phosphate; and (3) Bi-212 chloride. The three celllines that were used are V-79, which is a Chinese hamster lungfibroblast, Ehrlich-Lettre Ascites carcinoma (ATCC CCL 77), which is anascites producing -tumor, and NIH:OVCAR-3 (ATCC HTB 161), which is ahuman ovarian adenocarcinoma. These well known cell lines are predictiveof what happens in vivo. The cells were grown as monolayers and intospheroid formations and subsequently irradiated, as described herein.

The V-79 cells were maintained under exponential growth conditions inminimal essential medium (MEM) supplemented with 10 percent fetal bovineserum. The Ehrlich-Lettre Ascites carcinoma cells were maintained in 90percent NCTC 109 supplemented with 10 percent fetal bovine serum. TheNIH:OVCAR-3 cells were maintained in RPMI 1640 and 10 percent fetalbovine serum.

To initiate the formation of the spheroids, cells at a concentration ofabout 500,000 cells per flask were seeded onto petri dishes base-cookedwith 1 percent agar MEM without serum. After spheroids having a size ofabout 20 micrometers (μm) formed, the spheroids were transferred tospinner bottles and maintained for two to three weeks at a temperatureof 37° C. in a CO₂ incubator until they grew to 10 to 1000 μm. Prior toirradiation, the spheroids were sized and separated using a spheroidseparation column.

Cells from each of the cell lines were irradiated by subjecting thecells to X-ray radiation, P-32, and Bi-212. The X-ray source that wasused is a General Electric 250 kvp Maxitron, 26 milliAngstroms (mA) (HVL1.5 mm Cu) operated at a dose rate of 1.11 Greys per minute (Gy/min).The cells, in both monolayer and spheroid form were exposed at roomtemperature and then immediately plated for survival.

Cells in both monolayer and spheroid were exposed to P-32 chromicphosphate. The radiation dose to each sample was calculated as:

    D.sub.βt =73.8CE.sub.β T.sub.1/2 (1-e.sup.-(0.693t/T1/2)cGy,

where

D.sub.βt =dos at time t,

C=initial concentration of P-32 in microCuries per gram (μCi/gm) at thebeginning of time t,

E.sub.β =0.695 MeV, and

T_(2/3) =14.3 days (half-life of P-32).

After exposure, the cells were washed in phosphate-buffered saline (PBS)and immediately plated for survival.

The cell lines were also irradiated using Bi-212. Bismuth-212 was elutedfrom a generator as described in the aforementioned patent to Atcher etal., which was a cation-exchange column supporting Ra-224, with 1 ml of0.15 N HI, and the generator was purged with 2 ml of distilled deionizedwater. The acid was neutralized to pH 5 with 150 ml of 4 N sodiumacetate and sterilized by passage through a 0.22-μm Millex-GV filter. Insome experiments, 50 ml of 100 mM diethylenetriaminepentaacetic acid(DTPA) was added and the reaction mixture shaken before filtersterilization. The filter was purged with 1 ml of distilled deionizedwater, and the radioactivity was assayed using a calibrated 3×3-in. NaIdetector. The detector output was fed into a 4096 channel analyzer. The583-keV gamma from thallium-208 (Tl-208) was used to determine theBi-212 activity. The Tl-208 is a source that is traceable to theNational Bureau of Standards.

Cells were exposed over a period of four hours. The dose (D) permicroCurie was calculated and corrected for decay as follows: ##EQU1##where V=volume in milliliters, and

D in calculated in cGy/μCi-hr

The dose calculations were made presuming that the Bi-212 uniformlydistributed throughout the sample during the period of exposure.

Following plating, survival measurements were taken for each of theirradiated cell samples. The cells were first trypsinized. Between 100and 20,000 cells were placed in 10 milliliters (mL) of compete medium inpetri dishes. The plates were incubated at 37° C. for seven to ten days.The plates were then stained with crystal violet and colonies greaterthan 50 cells were scored. The surviving fraction was then determined.

Intrinsic radiosensitivity (D_(o)) was calculated from the survivalcurves. The mean lethal dose increment that was needed to reduce thesurviving population after treatment to 37 percent of the previous levelalong the straight line portion of the survival curve was used tocalculate D_(o). FIGS. 5 and 6 graphically illustrate the survivalcurves for irradiated cell lines of V-79, Ehrlich-Lettre Ascitescarcinoma and OVCAR-3 in monolayer and spheroid cell formations.Relative biological effectiveness (RBE) was determined as the ratio ofthe absorbed dose of X-ray to that of alpha radiation to produce thesame degree of biological effect. Table 5, below, shows a comparison ofthe radiosensitivities and relative biological effectiveness values thatwere calculated based upon the cell samples studied.

                  TABLE 5                                                         ______________________________________                                        COMPARISON OF RADIOSENSITIVITY (D.sub.0) AND                                  RELATIVE BIOLOGICAL EFFECTIVENESS (RBE)                                              D.sub.0                                                                              D.sub.0          D.sub.0                                               X-RAY  P-32     RBE     Bi-212 RBE                                            (in Gy)                                                                              (in Gy)  P-32    (in Gy)                                                                              Bi-212                                  ______________________________________                                        MONOLAYER                                                                     OVCAR-3  1.50     1.40     1.43  0.75   3.19                                  V-79     1.55     1.25     1.63  0.77   3.02                                  EHRLICH  --       --       --    0.65   --                                    SPHEROIDS                                                                     OVCAR-3  1.1      1.1      1.07  0.55   2.81                                  ______________________________________                                    

As can be seen from an examination of the data in Table 5, cells ofHIV:OVCAR-3 in both monolayer and spheroid form, and cells of V-79 inmonolayer form that were irradiated with Bi-212 showed considerablyhigher relative biological effects than those cells that were exposed toX-rays and radiation from P-32. That is, the relative biologicaleffectiveness using Bi-212 was shown to be 3.19, 2.81 and 3.02.respectively, compared to X-ray irradiation.

Likewise, the dose required to achieve equal cell survival rates wassignificantly lower for those cells exposed to Bi-212 compared to thosecells exposed to X-rays and radiation from P-32. For example, the doserate to achieve a 37 percent survival rate for the NIH:OVCAR-3 cells inspheroid form was 0.55 Gy for Bi-212 compared to 1.1 Gy for X-ray andP-32 irradiation; that is, one-half of the dose required by X-ray andP-32 irradiation was needed to achieve the same survival rate for cellsexposed to Bi-212.

Various spheroid formation cell samples were examined using bothelectron microscopy and autoradiography. Samples were prepared forelectron microscopy by placing the cells in 2 percent glutaraldehyde and2 percent formaldehyde electron microscopy grade in phosphate bufferedsaline (PBS) at a pH of 7.4 for ten minutes at room temperature. Thespheroids were then transferred to glass vials and placed in an ice bathfor ten minutes. Subsequently, the spheroid samples were washed with 0.2M sucrose in 0.1 M PO₄, fixed in 1 percent OsO₄ in 0.1 M phosphate andgently mixed for two hours as 4° C. After 24 hours, the cell sampleswere dehydrated with alcohol, fixed in upon and scanned by electronmicroscopy.

Spheroid cell samples that were exposed to Bi-212 were prepared forautoradiography after a two hour exposure to Bi-212. The samples werewashed with PBS, frozen, dehydrated, fixed in acetone and sectioned.Thin-layer sections were dipped in NTB-3 emulsion (commerciallyavailable from Kodak Corp. of Rochester, N.Y.), diluted 1:1 withdistilled water and heated to 42-44° C. for 10-20 minutes. The cellsamples were then developed in D1 developer (also available from KodakCorp.), stained with hematoxylin and eosin and examined usingautoradiography.

EXAMPLE 2 In Vivo Studies

In vivo studies were conducted to determine the distribution of Bi-212produced using the above-noted process and to compare the Bi-212distribution to the distribution of Pb-212 ferrous hydroxide incolloidal form. The in vivo studies were conducted using white, femaleNew Zealand rabbits weighing about 4.5 kilograms (about 2 pounds) each.Studies were also conducted using rabbits to determine the toxicitylevels, e.g., the maximum tolerated dose. Efficacy studies wereconducted using 25 gram (gm) female Swiss-Webster mice inoculated withEhrlich-Lettre Ascites carcinoma cells. The results of these studies arepresented below.

Distribution Studies

The rabbits were first anesthetized by intravenous titration. Apolyethylene catheter was then inserted into the lower right quadrant ofthe rabbits' abdominal cavities and a trace amount of technetium-99mpertechnetate was instilled to confirm that the catheter was correctlyplaced.

Groups of rabbits were instilled with a varying amounts (correlating tovarying activities) of Bi-212 that were prepared in accordance with thebefore-described process, neutralized to pH values of between 5.0 and7.4, and had added thereto a quantity of normal saline to make a 200 ccsample. To compare the distribution of Bi-212, Pb-212 ferrous hydroxidewas prepared in accordance with known methods. Rotmensch et al.,Gynecol. Oncol., 35:297-300 (1989). Groups of rabbits were alsoinstilled with the Pb-212 ferrous hydroxide preparation.

After instillation, the rabbits were rotated and imaged with a gammacamera set at 2.6 MeV. Where localization of the nuclide was identifiedin the rabbits, the rabbits were sacrificed and the localized area wasexcised and photographed.

Of the groups of rabbits instilled with the Bi-212 solution, a rabbitwas imaged and necropsied at each of one-half hour, one hour and threehours after instillation. The percent injected activity (%IA) in eachorgan was measured to determine the distribution of the Bi-212(Table 6).To determine whether chelating to DTPA improved the retention of Bi-212in the peritoneal fluid, a group of rabbits was instilled with Bi-212DTPA, and was imaged and necropsied at three hours followinginstillation. The percent activity remaining in the peritoneal fluid wasmeasured. The results of this comparison are shown in Table 7.

It was observed that the distribution of Bi-212 was relatively even whencompared to the non-uniform distribution of Pb-212 ferrous hydroxide. Atnecropsy of the rabbits that were instilled with the Pb-212 preparation,clumps of iron particles were found on peritoneal and bowel surfaces.

Imaging of the rabbits that were instilled with Bi-212 shows noclumping; i.e., no localized activity in the peritoneal cavity up to 3hours after instillation. Rabbits that were necropsied at one-half hour,one hour and 3 hours after instillation showed that 85.1 percent to 88percent of the activity remained in the peritoneal fluid rather thanaccumulating in various organs. Table 6, below, shows the distributionof Bi-212 in percent injected activity at one-half hour, one hour and 3hours after instillation. Surprisingly, as shown in Table 7, anexamination of the rabbits that were instilled with the Bi-212 DTPAshowed that only 51 percent of the activity remained in the peritonealcavity at 3 hours after instillation.

                                      TABLE 6                                     __________________________________________________________________________    BIODISTRUBTION OF Bi-212 AT 1/2, 1 AND 3 HOURS AFTER                          INTRAPERITONEAL INSTILLATION                                                          1/2 HOUR (% IA)                                                                         1 HOUR (% IA)                                                                           HOUR (% IA)                                       ORGAN   #1 #2  #3 #1 #2  #3 #1 #2 #3                                          __________________________________________________________________________    PER. FLUID                                                                            85.0                                                                             70.3                                                                              90.1                                                                             85.7                                                                             85.1                                                                              88 75.8                                                                             80.2                                                                             83.8                                        LIVER   0.68                                                                             0.63                                                                              0.18                                                                             1.45                                                                             0.41                                                                              0.23                                                                             2.26                                                                             2.25                                                                             1.17                                        KIDNEY  0.74                                                                             0.98                                                                              0.19                                                                             1.59                                                                             0.53                                                                              0.34                                                                             2.15                                                                             1.21                                                                             2.10                                        REPR.   0.63                                                                             0.09                                                                              0.09                                                                             0.40                                                                             0.04                                                                              0.08                                                                             0.53                                                                             0.61                                                                             0.59                                        STOM.   -- 0.26                                                                              0.34                                                                             -- --  0.09                                                                             0.90                                                                             0.76                                                                             0.59                                        LOWER   3.36                                                                             1.90                                                                              0.53                                                                             -- --  0.87                                                                             7.14                                                                             5.87                                                                             9.14                                        INTES.                                                                        & COLON                                                                       UP. SM. 4.35                                                                             2.16                                                                              0.87                                                                             5.24                                                                             2.28                                                                              0.96                                                                             8.04                                                                             6.63                                                                             9.73                                        INTES.                                                                        CIRC.   1.23                                                                             --  0.22                                                                             2.22                                                                             1.17                                                                              0.44                                                                             3.4                                                                              1.86                                                                             2.96                                        BLOOD                                                                         RED MARROW                                                                            0.05                                                                             0.10                                                                              0.03                                                                             -- --  -- 0.20                                                                             0.20                                                                             0.09                                        CARC.   15.3                                                                             5.90                                                                              1.33                                                                             40.2                                                                             1.70                                                                              2.17                                                                             8.70                                                                             4.01                                                                             4.89                                        VOL. PER.                                                                             183                                                                              168 184                                                                              142                                                                              162 167                                                                              188                                                                              200                                                                              192                                         FL.                                                                           RECOVER                                                                       (ml)*                                                                         __________________________________________________________________________     *Initial volume = 200 ml                                                      (Per  peritoneal; Repr.  reproductive; Stom.  stomach; Intes.  intestine;     Up Sm.  upper small; Circ.  circulating; Carc.  carcass)                      IA = injected activity                                                   

                  TABLE 7                                                         ______________________________________                                        COMPARISON OF RETENTION OF CHELATED AND                                       NON-CHELATED Bi-212 IN PERITONEAL FLUID                                               % ACTIVITY IN                                                                           % ACTIVITY                                                          PERITONEAL                                                                              IN BLOOD    % ACTIVITY                                              FLUID     VOLUME      IN URINE                                        ______________________________________                                        Bi-212    80          4.5         6.0                                         Bi-212-DTPA                                                                             51          9.4         40                                          ______________________________________                                    

Toxicity Studies

The peritoneal cavities of rabbits were instilled with graded doses ofBi-212. The rabbits were followed until death or for up to three monthsafter their blood counts normalized. The rabbits were necropsied atdeath or termination of the study and their organs were microscopicallyexamined to determine the effect of the Bi-212 on healthy organs.

It was observed that the maximum dose of Bi-212 tolerated by the rabbitswas 60 mCi. At 60 mCi, microscopic examination of organs three monthsafter instillation showed only mild blunting of intestinal villi. Dosesgreater the 60 mCi caused death within three days. At 80 mCi, therabbits survived about three days and exhibited marked individual cellnecrosis of gland epithelium of the small and large intestines. At adose of 100 mCi, the rabbits survived about three days and exhibiteddiffuse epithelial necrosis.

Efficacy Studies

Efficacy studies were conducted using groups of five 25 gm femaleSwiss-Webster mice that were intraperitoneally inoculated with 10⁶Ehrlich-Lettre Ascites carcinoma cells. The mice were subsequentlyinstilled with 100 μCi of Bi-212 in up to 1 cc normal saline 48 hoursafter inoculation with the Ehrlich-Lettre Ascites carcinoma cells. Themice were sacrificed at one-half hour, one hour and 3 hours afterinstillation with the Bi-212 solution. Control groups were inoculatedwith the Ehrlich-Lettre Ascites carcinoma cells only.

It was observed that the maximum tolerated dose in the mice was 0.65mCi. As shown in Table 8, below, necropsying the mice at one-half hour,one hour and 3 hours after instillation of the Bi-212 solution showedthat 61.4 percent of the activity remained in the mice at 3 hours afterinstillation. Examination of mice in the control group revealed thatsolid nests of tumor cells formed and infiltrated the tissue within 48hours after inoculation.

The mice in the control group died within 21 days. Mice that weretreated with Bi-212 had a median survival time of 82 days aftertreatment. In 40 percent of the mice, there is a cure with no evidenceof disease 3 months later. The results are illustrated graphically inFIG. 7.

                  TABLE 8                                                         ______________________________________                                        RETENTION OF Bi-212 AFTER INTRAPERITONEAL INJECTION                           IN MICE                                                                       SACRIFICE    CARCASS     URINE     STOOL                                      TIME (MIN)   % IA        % IA      % IA                                       ______________________________________                                        1            88.3        0.025     --                                         10           87.3        2.2       --                                         16           66.8        2.9       0.20                                       30           79.1        6.9       0.53                                       61           68.5        15.9      1.3                                        120          62.9        19.5      6.7                                        180          61.4        24.8      8.3                                        ______________________________________                                    

Based upon the in vivo studies, a contemplated dose of 100 mCi to ahuman produces an alpha and gamma dose to any organ of less than 150cGy. The calculated alpha dose to the gastrointestinal tract, liver andkidneys is 87.7, 24.6 and 92.5 cGy, respectively, and the calculatedgamma dose is 7.39, 4.14 and 5.00 cGy, respectively. Table 9, below,provides the estimated total dose in humans based upon an initial doseof 100 mCi of Bi-212.

                  TABLE 9                                                         ______________________________________                                        ESTIMATED TOTAL DOSE IN HUMANS/100 mCi OF Bi-212                                         GAMMA DOSE/  ALPHA DOSE/  TOTAL                                    ORGAN      100 mCi      100 mCi      DOSE                                     ______________________________________                                        RED MARROW 2.30         2.45         4.75                                     BRAIN      0.038        2.45         2.49                                     BREASTS    0.643        2.45         3.09                                     GI TRACT   7.39         87.7         95.1                                     KIDNEYS    5.00         92.5         97.5                                     LIVER      4.14         24.6         28.7                                     LUNGS      1.33         2.45         3.78                                     OVARIES    5.42         102.4        107.8                                    PANCREAS   17.2         2.45         19.7                                     THYROID    0.154        2.45         2.60                                     UTERUS     21.2         102.4        123.6                                    REMAINDER  1.78         2.45         4.23                                     TOTAL BODY 1.83         2.45         4.28                                     ______________________________________                                    

From the foregoing it will be observed that numerous modifications andvariations can be effectuated without departing from the true spirit andscope of the novel concepts of the present invention. It is to beunderstood that no limitation with respect to the specific embodimentsillustrated is intended or should be inferred. The disclosure isintended to cover by the appended claims all such modifications as fallwithin the scope of the claims.

What is claimed is:
 1. A process for producing substantiallyradio-impurity-free Bi-212 comprising the steps of:(a) contacting anacidic Pb-212 feed solution with an extraction medium having a pluralityof binding sites thereon adapted to bind said Pb-212 thereto, to form aPb-212-laden extraction medium and less strongly bound contaminants; (b)rinsing said Pb-212-laden extraction medium with a second acid solutionto remove the less strongly bond contaminants therefrom and to form asubstantially impurity-free Pb-212-laden extraction medium; (c)maintaining said substantially radio-impurity-free Pb-212-ladenextraction medium for a predetermined period of time so as to formBi-212 from said Pb-212 by radioactive decay; (d) introducing a thirdacid solution to said substantially impurity-free Pb-212-ladenextraction medium to release said Bi-212 therefrom and form a Bi-212acid solution; and (e) eluting said Bi-212 acid solution from saidsubstantially impurity free Pb-212-laden extraction medium to form asubstantially radio-impurity free Bi-212 acid solution.
 2. The processfor producing substantially impurity-free Bi-212 in accordance withclaim 1 including contacting said substantially radio-impurity-freeBi-212 acid solution of step (e) with a second extraction medium havinga plurality of binding sites thereon adapted to bind Pb-212 thereto. 3.The process for producing substantially impurity-free Bi-212 inaccordance with claim 1 including eluting said Bi-212 from saidsubstantially impurity free Pb-212-laden extraction medium at intervalsof about two hours.
 4. The process for producing substantiallyimpurity-free Bi-212 in accordance with claim 1 including neutralizingsaid substantially radio-impurity free Bi-212 acid solution.
 5. Theprocess for producing substantially impurity-free Bi-212 in accordancewith claim 1 wherein said Pb-212 acidic feed solution is a first acidsolution containing Pb-212 or a Pb-212 generating material.
 6. Theprocess for producing substantially impurity-free Bi-212 in accordancewith claim 1 wherein said first acid solution has a concentration ofabout 0.5 N to about 4.0 N.
 7. The process for producing substantiallyimpurity-free Bi-212 in accordance with claim 1 wherein said second acidsolution has a concentration of about 0.5 N to about 4.0 N.
 8. Theprocess for producing substantially impurity-free Bi-212 in accordancewith claim 1 wherein said third acid solution has a concentration ofabout 0.5 N to about 1.5 N.
 9. The process for producing substantiallyimpurity-free Bi-212 in accordance with claim 1 wherein said extractionmedium is a solid phase-supported extractant.
 10. A process forproducing substantially radio-impurity-free Bi-212 comprising the stepsof:(a) contacting an acidic Pb-212 feed solution with a solidphase-supported extractant having a plurality of binding sites thereonadapted to bind said Pb-212 thereto, to form a Pb-212-laden extractantand less strongly bound contaminants; (b) rinsing said Pb-212-ladenextractant medium with a second acid solution to remove the lessstrongly bound contaminants therefrom and to form a substantiallyimpurity-free Pb-212-laden extractant; (c) maintaining saidsubstantially radio-impurity-free Pb-212-laden extractant for apredetermined period of time so as to form Bi-212 from said Pb-212 byradioactive decay; (d) introducing a third acid solution to saidsubstantially impurity-free Pb-212-laden extractant medium to releasesaid Bi-212 therefrom and form a Bi-212 acid solution; and (e) elutingsaid Bi-212 acid solution from said substantially impurity freePb-212-laden extractant to form a substantially radio-impurity freeBi-212 acid solution.
 11. An apparatus for producing substantiallyradio-impurity-free Bi-212 from a starting material having Pb-212 or aPb-212 generating material comprising:an extraction medium having aplurality of binding sites thereon, said binding sites having anaffinity for binding Pb-212 thereto and having a lower affinity forbinding Bi-212 thereto; a first acid supply in flow communication withsaid extraction medium, said acid supply adapted to supply a first acidto carry the Pb-212 to said extraction medium; a first vessel adapted toretain said extraction medium and further adapted to maintain contactbetween said extraction medium and said acid; a second acid supply inflow communication with said vessel, said acid supply adapted to supplya second acid to said vessel; a mixing chamber in flow communicationwith said vessel adapted to receive a liquid solution therefrom, saidmixing chamber having a plurality of input means connected thereto; anda discharge line in flow communication with said mixing chamber.
 12. Theapparatus in accordance with claim 11 further including a second vesseladapted to retain a quantity of extraction medium therein and furtheradapted to maintain contact between said extraction medium and saidacid, said second vessel being in flow communication with said firstvessel and in flow communication with said mixing chamber.
 13. Theapparatus according to claim 11 wherein said apparatus includes a pairof first vessels wherein only one of the pair of first vessels isoperable at a time.
 14. The apparatus according to claim 11 including aplurality remotely actuated valves therein.
 15. The apparatus accordingto claim 11 wherein said apparatus includes a solid phase-supportedextractant.
 16. A process for treating target cells that comprisescontacting the target cells with a biologically effective amount of apharmaceutically acceptable composition comprising substantiallyimpurity-free Bi-212 in solution.
 17. The process in accordance withclaim 16 wherein the Bi-212 is uncompleted.
 18. The process inaccordance with claim 16 wherein the target cells are a microscopiccarcinoma.
 19. The process in accordance with claim 16 wherein thetarget cells are within a host mammal.
 20. The process in accordancewith claim 19 wherein the Bi-212 is administered intraperitoneally tocontact the target cells.
 21. A pharmaceutical composition for thetreatment of carcinoma comprising a biologically effective amount ofsubstantially radio-impurity free Bi-212.
 22. The pharmaceuticalcomposition in accordance with claim 21 wherein the Bi-212 isuncomplexed.
 23. The pharmaceutical composition in accordance with claim21 wherein the carcinoma is a microscopic carcinoma.
 24. Thepharmaceutical composition in accordance with claim 21 that isadministered intraperitoneally.